Therapeutic polypeptides and proteins can be expressed in a variety of host cells including bacterial cells, E. coli cells, fungal or yeast cells, cells of a microorganism, insect cells, and mammalian cells. Fungal hosts such as the methylotrophic yeast Pichia pastoris has distinct advantages for therapeutic protein expression—e.g. it does not secrete high amounts of endogenous proteins, it has a strong inducible promoter, it can be grown in defined chemical media, and it can produce high titers of recombinant proteins (Cregg et al., Mol. Biotech. 16:23-52 (2000)). Yeast and filamentous fungi have both been successfully used for the production of recombinant proteins, both intracellular and secreted (Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1): 45 66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M., et al. 1992 Abstr. Papers Amer. Chem. Soc. 203: 121-BIOT; Svetina, M., et al. 2000 J. Biotechnol. 76(23): 245-251. Pichia is a remarkable host cell for expression of recombinant human serum albumin (HSA). However, the expression of other therapeutic polypeptides including polypeptides genetically fused with HSA faces the technical barriers of undesired proteolysis and glycosylation.
Heterologous proteins expressed in P. pastoris may contain additional mannose sugars resulting in “high mannose” glycans, as well as mannosylphosphate groups which impart a negative charge onto a protein. Glycosylated proteins with either high mannose glycans or charged mannans are a high risk for eliciting an immune response in humans (Takeuchi, Trends in Glycosci. & Glycotech., 9:S29-S35 (1997); Rosenfeld et al., J. Biol. Chem., 249:2319-2321 (1974)). Accordingly, it is desirable to produce therapeutic peptides, polypeptides and/or proteins in fungal host systems, such that the pattern of glycosylation is identical or at least similar to that in humans.
Thus, there is a need for yeast strains, in particular Pichia strains that are capable of producing heterologous peptides, polypeptides and/or proteins with reduced proteolysis and/or glycosylation when compared with wild type strains. In addition, there is a need to identify genes within yeast strains, in particular Pichia strains, responsible for producing proteins involved in proteolytic and glycosylation pathways.